The present invention relates to a process for the preparation of derivatives of Ruscus Aculeatus steroid glycosides (ruscosaponins).
More particularly, the invention relates to the preparation of desglucodesrhamnoruscin by hydrolysis of ruscoside and/or desglucoruscoside through the fermentation route.
Desglucorhamnoruscin and the corresponding free aglycons, ruscogenin and neoruscogenin, which can be easily obtained from desglucodesrhamnoruscin by acid hydrolysis, are valuable pharmaceutical active principals having antiinflammatory and connective-protecting activities.
The chemical preparation of said active principles starting from ruscoside or desglucoruscoside is however problematic, since it requires drastic conditions, such as hydrolysis with strong acids, and complex operative steps, which yield a very heterogeneous mixture of intermediates and products.
It would therefore be highly desirable to provide a process for the preparation of desglucodesrhamnoruscin, which overcomes the drawbacks mentioned above connected with the known chemical processes.
The present invention meets such a need, by providing a process for the preparation of desglucodesrhamnoruscin which comprises the hydrolysis of Ruscus aculeatus steroid glycosides (ruscosaponins) through fermentation of a substrate containing said glycosides by means of fungi of the aspergillus niger species.
A culture broth is typically used as a nutrient complex substrate.
Fermentation is generally carried out at a temperature of 25-30xc2x0 C., preferably 27xc2x0 C., under stirring and aeration so as to attain a pO2 higher than or equal to 50%.
The concentration of the starting steroid glycosides usually ranges from 5 to 15% w/v, preferably from 8 to 10% w/v and the pH of the culture broth ranges from 4 to 6, preferably 4.5-5.5.
The biotechnological process of the present invention allows to carry out the whole reactions sequence in a single fermentation step, in that the microorganism, selected with suitable microbiological techniques, is capable of expressing the necessary enzymatic activities for operating all the transformation sequential reactions, from the starting complex heteroglycoside to the monoglycoside or the final aglycone. Said hydrolase transformations comprise in fact a sequence of xcex2-glucosidase, xcex1-rhamnosidase reactions on the intermediates which are successively released during the process. A further xcex1-arabinosidase reaction allows to obtain the free aglycone (ruscogenin).
Said approach is quite novel, in that no applicative examples of said procedure for the preparation of said products can be found in literature.
The microorganisms suitable for carrying out the transformations involved are obtained by selection on synthetic or semi-synthetic media, added with the same substrates to be transformed, in addition to or in place of the conventional carbon sources (glucose, saccharose, and the like). The concerned substrates (ruscoside, desglucoruscoside) can be added in this case in even high concentrations, e.g. 90-100 g/L. The agarized isolation media comprise the usual formulations for microbiology, such as Malt Agar and Czapek Agar, or similar formulations, wherein the nitrogen source is represented by peptones, urea, ammonium nitrate, and the like, whereas the conventional carbon source (glucose, saccharose) has been substituted or supplemented by ruscoside or desglucoruscoside. Said media can be further added with mineral salts of potassium, magnesium, manganese, zinc etc., such as phosphates, sulfates and/or chlorides. The pH of the isolation media can range from 4 to 6, preferably from 4.5 to 5.5.
The microorganisms suitable to the required biotrasformations are recovered by scalar dilution and plating of aqueous suspensions of samples of soil, humus, vegetable extracts and other similar organic sources.
The microbial cultures selected as described above are isolated in microbiology test-tubes containing the same culture media and used for the biotransformation of ruscoside and desglucoruscoside, added in high concentrations (to 100 g/L) to liquid culture media containing the same nitrogen sources as used in the isolation media, such as urea or peptone, with the addition of phosphates and other mineral salts, as described above, at pH ranging from 4 to 6, preferably 4.5÷5.5.
Following the procedures described it has been found that selected cultures of Aspergillus niger are capable of transforming ruscoside and desglucoruscoside into desglucodesrhamnoruscin, a direct precursor of ruscogenins, by a sequence of enzymatic xcex2-glucosidase and xcex1-rhamnosidase reactions.
A subsequent xcex1-arabinosidase reaction provides the saponin in the aglyconic form (ruscogenin-neoruscogenin).
The selected culture is capable of operating said transformations growing in controlled (thermostatic) conditions, at optimal temperatures ranging from 25xc2x0 C. to 30xc2x0 C., under stirring on a rotatory shaker (200÷300 rpm). Said fermentation can also be carried out in a suitable bioreactor, at different scale levels, for the industrial production of the desired saponin derivatives.
The microorganisms used for said biotransformation are capable of steadily maintaining the catalytic activity, even for repeated fermentation cycles, in batch or continuous processes.
The present process provides important advantages, such as less complex the steps for the separation and recovery of the product, and is also easy to carry out as well as cost-saving.
The selected microorganisms can be frozen for the to long-term storage, in suspensions enriched with cryopreservatives, such as glycerol, peptone and the like, at temperatures ranging from xe2x88x9280xc2x0 C. to xe2x88x92196xc2x0 C. (in liquid nitrogen), or subjected to freeze-drying treatments.
The progress of the bioconversion can be monitored by TLC and HPLC analysis on the culture broth, using the following analytical methods:
TLC Analysis
Silica gel plates 60 F250 Merck
Eluents:
A) Ethyl acetate-Methanol 9:1
B) Ethyl acetate-Methanol-Water 100:15:10.
Detection: reaction with 10% sulfuric acid and heating to 120xc2x0 C. for 5 minutes, then visible and UV detection.
HPLC Analysis
Column: Supelcosil LC18, 250xc3x974,6 mm, 5 xcexcm
Eluent: acetonitrile-water 60:40
Wavelength: 200 nm
Injection volume: 10 xcexcl
Flow: 1 mL/min.
The final biotransformation products, such as desglucodesrhamnoruscin, can be recovered by extraction of the culture broth with n-butanol, subsequent purification steps with chlorinated solvents (such as trichloroethane) and silica gel chromatography. Finally, the product can be crystallized from different solvents, such as isopropanol, ethyl acetate, chloroform, acetone, methanol. In addition to the main product, desglucodesrhamnoruscins esterified at C-2xe2x80x2, for example with 2-hydroxy-3-methylpentanoic acid, can be obtained.
The saponins in the aglyconic form (ruscogenins) are obtained by acid hydrolysis of the fermentation products described above.
The following examples disclose the invention in greater detail.